Covalent inhibition of the SARS-CoV-2 NiRAN domain via an active-site cysteine

Abstract

The kinase-like nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain of nsp12 in SARS-CoV-2 catalyzes the formation of the 5' RNA cap structure. This activity is required for viral replication, offering a new target for the development of antivirals. Here, we develop a high-throughput assay to screen for small molecule inhibitors targeting the SARS-CoV-2 NiRAN domain. We identified NiRAN covalent inhibitor 2 (NCI-2), a compound with a reactive chloromethyl group that covalently binds to an active site cysteine (Cys53) in the NiRAN domain, inhibiting its activity. NCI-2 can enter cells, bind to, and inactivate ectopically expressed nsp12. A cryo-EM reconstruction of the SARS-CoV-2 replication-transcription complex bound to NCI-2 offers a detailed structural blueprint for rational drug design. Although NCI-2 showed limited potency against SARS-CoV-2 replication in cells, our work lays the groundwork for developing more potent and selective inhibitors targeting the NiRAN domain. This approach presents a promising therapeutic strategy for effectively combating COVID-19 and potentially mitigating future coronavirus outbreaks.

Keywords: COVID-19; NiRAN; RNA capping; SARS-CoV-2; drug discovery; drug screening; enzyme inhibitor; enzyme structure; inhibitor; pseudokinase.

Figure 1

A high-throughput screen (HTS) identifies inhibitors of the NiRAN domain.A, schematic representation of the catalytic reaction quantified in the HTS used in this study to identify inhibitors of NiRAN-dependent nsp9 AMPylation. B, dependence of nsp12 concentration on ATP consumption (50 μM) during NiRAN-mediated AMPylation of nsp9 (50 μM). The red point indicates the concentration of nsp12 used in the HTS. C, rank plot depicting the range of inhibition by compounds of NiRAN-catalyzed AMPylation of nsp9 in the screen. Mean (n = 3 for technical replicates) differential scanning fluorimetry profiles of nsp12 (10 μM) in the presence of NCI-1 (20 μM) (D) and NCI-2 (20 μM) (E). Chemical structures of NCI-1 (F) and NCI-2 (G). Dose-response curves of nsp12-dependent AMPylation of nsp9 with NCI-1 (H) or NCI-2 (I). The assay contained 100 nM nsp12, 50 μM nsp9, and 50 μM ATP. The reaction products were quantified via Kinase-Glo Plus. The IC₅₀ values are shown (n = 4 for technical replicates). Error bars represent the standard error of the mean (SEM). NCI-1, NiRAN covalent inhibitor 1; NiRAN, nidovirus RdRp-associated nucleotidyl transferase.

Figure 2

NCI-1 and NCI-2 form covalent bonds with Cys53 within the SARS-CoV-2 NiRAN domain. Intact mass LC-MS spectra of nsp12 (black trace) overlayed with nsp12 incubated with NCI-1 (red trace, A) or NCI-2 (red trace, B) at a 1:1.5 protein:drug ratio. The observed masses are shown above the respective peaks for each curve. The theoretical mass for nsp12 is 106,660.24 Da. The theoretical mass shifts (Δm) for NCI-1 and NCI-2 bound to nsp12 are 191.53 Da and 204.57 Da, respectively. MS/MS spectra of nsp12 depicting the peptide ion DKVAGFAKFLKTNCCRFQEK with covalent attachment of either NCI-1 (+192 Da) (C) or NCI-2 (+205 Da) (D) on residue C53 of nsp12. Spectra were generated via higher-energy collisional dissociation (HCD) and fragment ions shown in red depict mass shifts corresponding to the addition of the modifying group. The precursor ion is labeled with an asterisk. Any fragment ions labeled with (∗) correspond to loss of H₂O (−18 Da) or NH₃ (−17 Da). E, sequence logo highlighting the conservation of C53 in the NiRAN domain. The heights of the amino acid stacks correspond to the sequence conservation at given positions. Intact mass LC/MS spectra of nsp12^C53A (black) overlayed with nsp12^C53A incubated with NCI-1 (red trace, F) or NCI-2 (red trace, G). The observed masses are shown above the respective peaks for each curve. Intact mass LC/MS spectra of nsp12^C53T (black trace) overlayed with nsp12^C53T incubated with NCI-1 (red trace, H) or NCI-2 (red trace, I). The observed masses are shown above the respective peaks for each curve. Intact mass LC-MS spectra of SARS-CoV-1 nsp12 (black trace, J), MERS nsp12 (black trace, K), and human SelO^U667C (black trace, L) overlayed NCI-2 (red traces). The observed masses are shown above the respective peaks for each curve. NCI-1, NiRAN covalent inhibitor 1; NiRAN, nidovirus RdRp-associated nucleotidyl transferase; MERS, Middle East respiratory syndrome.

Figure 3

Cys53 within the NiRAN domain is essential for the inhibitory activity of NCI-2.A, AMPylation activity of nsp12, nsp12^C53T, or nsp12^C53A (100 nM) in the presence of NCI-2 (20 μM). Reactions were allowed to proceed for 30 min, and the products were analyzed by Kinase-Glo Plus (n = 4 for technical replicates; error bars indicate SEM; ∗∗∗∗p < 0.0001 from 2-way ANOVA with Tukey post hoc multiple comparisons for the indicated group compared against all other groups except the no enzyme control group). B, time-dependent RNAylation of nsp9 (20 μM) by nsp12, nsp12^C53T, or nsp12^C53A (500 nM). Reactions were initiated with 100 μM of SARS-CoV-2 genomic leader sequence corresponding to the first 10 bases in the genome, and the reaction products were analyzed by SDS-PAGE and Coomassie staining. C, RNAylation activity of nsp12, nsp12^C53T, or nsp12^C53A (500 nM) following incubation with NCI-2 (20 μM) for 30 min prior to the start of the reaction. Reaction products were analyzed as in (B). D, nsp12, nsp12^C53T, or nsp12^C53A (500 nM)-mediated deRNAylation of nsp9 (20 μM) following incubation with NCI-2 (20 μM) for 30 min prior to the start of the reaction. Reaction products were analyzed as in (B). Note that the deRNAylation of nsp9 is a readout of core cap GpppA formation (23). E, nsp12, nsp12^C53T, or nsp12^C53A (500 nM) RdRp activity in an RNA extension activity. nsp12 and mutants were preincubated with the cofactors nsp7 (5 μM), nsp8 (5 μM), and NCI-2 (20 μM) for 30 min, initiated by the addition of a fluorescently labeled self-priming hairpin RNA and NTPs. The reaction products were separated by Urea-PAGE and analyzed by fluorimetry. NCI, NiRAN covalent inhibitor; NiRAN, nidovirus RdRp-associated nucleotidyl transferase; RdRp, RNA-dependent RNA polymerase.

Figure 4

NCI-2 enters cells, inhibits ectopically expressed nsp12, and shows limited potency against SARS-CoV-2.A, LDH release assay as a readout of cell toxicity following treatment of HEK293A cells with NCI-2 for 8 h at 37 ^oC and 5% CO₂, with vehicle (1% DMSO) treated cells as a negative control and lysed cells as positive control. B, intracellular accumulation of NCI-2 in cells at 4 and 8 h post treatment. HEK293A cells cultured in serum-free medium were incubated with NCI-2 (80 μM) for 30 min, and NCI-2 was quantified by LC-MS/MS analysis. C, chemical structure of the NCI-2 derivative NCI-3 depicting the alkyne handle (red). D, dose-response curve of nsp12-dependent AMPylation of nsp9 with NCI-3. Reactions were analyzed as in Figure 1H. The IC₅₀ value is shown. E, LDH release assay as a readout of cell toxicity following treatment of HEK293A cells with NCI-3 at different concentrations. F, analysis of HEK293A cell lysates for biotinylation via avidin-HRP or nsp12 via protein immunoblotting following treatment with NCI-3 (80 μM). Following 2- and 8-h incubations, cells were harvested, and the lysates underwent a copper-catalyzed click reaction with biotin azide to generate biotin-NCI-3 conjugates for detection. Actin is shown as a loading control. G, RNAylation activity of Flag-tagged nsp12 (Flag-nsp12) immunoprecipitated from HEK293A lysates following treatment of the cells with 80 μM NCI-2 (in cellulo). Purified nsp12 (500 nM) was also included in these assays as a control and treated with NCI-2 (80 μM) for 30 min prior to the start of the reaction (in vitro). Reaction products were analyzed as in Figure 3B. Infectivity of SARS-CoV-2-Wu-1-zsGreen in the presence of varying concentrations of remdesivir (H) or NCI-2 (I). A549-TMPRSS2-ACE2 cells were infected with SARS-CoV-2 for 30 min at 37 °C. Cells were washed to remove virus and medium containing DMSO and the indicated drugs were added back. At 7 h post infection, cells were fixed with 4% PFA. Viral infectivity was quantified by flow cytometry. N = 3 biological replicates. Statistical analysis was determined by paired t test. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001, ns, not significant. DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; LC-MS/MS, liquid chromatography with tandem mass spectrometry; LDH. lactate dehydrogenase; NCI, NiRAN covalent inhibitor; PFA, paraformaldehyde.

Figure 5

Structure-activity analysis of NCI-2.A, kinetic analysis curve of a time- and dose-dependent assay with calculated values for k_inact, K_i, and k_inact/K_i for NiRAN-dependent nsp9 AMPylation inhibition activity with 100 nM nsp12 measured via Kinase-Glo Plus (n = 4). B, chemical structure of NCI-2 with indicated IC₅₀ values for inhibition of nsp9 AMPylation. NCI-2 derivatives with the chloromethyl (C) hydroxyl (D) or methyl ester (E) functional groups substituted to the functional group indicated in red for each structure. IC₅₀ values quantified as in Figure 1I are shown. NCI, NiRAN covalent inhibitor; NiRAN, nidovirus RdRp-associated nucleotidyl transferase.

Figure 6

Cryo-EM structure of NCI-2 bound to Cys53 in the NiRAN domain.A, cartoon representation of the NiRAN domain active site bound to the covalent inhibitor NCI-2 with the coulombic density presented as a transparent surface. Superimposition of nucleotides bound in the NiRAN active site in “base-in” (B) and “base-up” (C) conformations (PDB ID 8SQ9). Cartoon representations of bound NCI-2 (D) and an apo structure of the NiRAN domain (E) (PDB: 7UO4) (34). Selected residues important for catalysis are shown as sticks. Coulombic density for the compound and the Cys53 is shown as mesh. Map was sharpened with a B-factor = −71.2. F, LigPlot (61) showing interactions between NCI-2 and the NiRAN domain. G, surface representation of the NiRAN active site proximal to the inhibitor, shown as sticks. NCI, NiRAN covalent inhibitor; NiRAN, nidovirus RdRp-associated nucleotidyl transferase; PDB, Protein Data Bank.